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研究生:黃鉑惠
研究生(外文):Bo-HuiHuang
論文名稱:CARD9的生化功能及結構分析
論文名稱(外文):Biochemical and structural studies on CARD9
指導教授:羅玉枝羅玉枝引用關係
指導教授(外文):Yu-Chih Lo
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技與產業科學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:78
中文關鍵詞:CARD9BCL10CBM複合物
外文關鍵詞:CARD9BCL10CBM complex
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當細胞接收到外界刺激,CARMA家族會與BCL10及MALT1形成CBM複合物來誘導NF-B的活化。在CARMA家族中,CARD9在結構上與其他成員較不同,缺乏維持不活化態的MAGUK。先前研究指出,CARD9參與抵抗真菌、細菌及病毒感染的機制。CARD9的缺陷會造成免疫系統異常,進而導致真菌感染,可推論CARD9在免疫系統中是重要的角色。然而,CARD9如何與BCL10、MALT1形成CBM複合物的機制尚不清楚。因此,本研究想藉由解出CARD9的蛋白質結構並利用生化方法來了解CARD9在NF-B路徑中的分子機制。首先藉由二級結構預測建構出不同長度的CARD9,再以親和性層析與分子篩色譜法進行純化。取得大量且純的CARD9蛋白質,透過負染,電子顯微鏡下觀察發現CARD9的粒子與背景有強烈的對比,利於後續數據的收集及計算。此外,將CARD9與不同長度的BCL10共同純化來取得蛋白質複合物。蛋白質體外結合實驗結果顯示在BCL10上的關鍵突變會加強與CARD9的結合親和力,並進一步取得CARD9與BCL10的蛋白質複合物。根據上述結果可以更了解CARD9的結構資訊以及在NF-B路徑中的分子特性。
When cell receives external stimuli, CARMA family recruits downstream components BCL10 as well as MALT1 to form a signaling complex called the CBM complex. The CBM complex induces NF-B signaling pathway against pathogen invasion. CARD9 is structurally dissimilar to other members in CARMA family, which lacks MAGUK domain for maintaining inactive state. Previous research showed that CARD9 involved in the antifungal, antibacterial as well as antiviral immune responses, and CARD9 deficiency leads to immune system disorders and fungal infections. It shows that CARD9 plays a crucial role in immune system. However, mechanism of how CARD9 forms the CBM complex with BCL10 and MALT1 is still unknown. Hence, I propose to solve the structure of CARD9 and use biochemical assays to reveal its molecular mechanism in NF-B pathway. Different lengths of CARD9 are constructed via secondary structure prediction and purified by the affinity chromatography and size exclusion chromatography. The pure and large quantity of CARD9 observed under electron microscope via the negative stain showed high contrast between the particle of CARD9 and background, which contributed to data collection and calculation. Besides, CARD9 co-purified with different lengths of BCL10 could form complex with BCL10. Furthermore, the result of the pull down assay showed that the key mutants on BCL10 enhanced binding affinity with CARD9, suggested that the protein complex could be further obtained. The result got from above experiments, it will help us to know the structural information and molecular characterization of CARD9 in NF-B pathway.
Chinese Abstract (中文摘要).....I
Abstract.....II
Acknowledgments.....V
Table of Contents.....VI
Contents of Tables.....VIII
Contents of Figures.....IX
Contents of Appendices.....XI
Abbreviation List.....XII
1.Research Background.....1
1-1 The NF-κB signaling pathway.....1
1-2 Caspase recruitment domain (CARD) and membrane-associated guanylate kinase-like domain (MAGUK) protein, CARMA family.....2
1-3 Caspase recruitment domain-containing protein 9 (CARD9).....4
1-4 The aim of this study.....5
2.Materials and Methods.....7
2-1 The preparation of competent cell.....7
2-2 Reconstruction of CARD9.....7
2-3 Mutagenesis of CARD9.....8
2-4 Protein expression constructs.....9
2-5 Recombinant protein expression and purification.....10
2-6 X-ray crystallography.....10
2-7 Negative stain electron microscopy (NS-EM).....11
2-8 Multiangle light scattering (MALS).....12
2-9 Pull down assay of CARD9 and BCL10.....12
3.Results.....13
3-1 Expression and purification of recombinant proteins.....13
3-2 The negative staining results of different lengths of CARD9 protein.....16
3-3 X-ray crystallography.....17
3-4 Multi Angle Light Scattering (MALS).....18
3-5 Co-purification of CARD9 and BCL10 to form complex.....19
3-6 CARD9 pulldown BCL10 mutants.....21
3-7 Mutagenesis CARD9 and pulldown BCL10 mutants.....22
4.Discussion.....24
References.....30
Tables.....35
Figures.....38
Appendices.....68
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